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Characterizing the proteomes of selected members of the Mycobacterium tuberculosis complex

Botha, Louise (2014-04)

Thesis (MScMedSC)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the
world’s population and causes approximately 2 million deaths per year. This pathogen is a
member of the Mycobacterium tuberculosis complex (MTBC) which constitutes eleven
members that share 99.9% similarity at nucleotide level and have near identical 16S rRNA.
MTBC members cause Tuberculosis in a variety of host species. M. bovis and M. caprae
form part of the animal-adapted MTBC members that cause disease in a variety of animal
hosts (primarily bovidae) and goats, respectively. Extensive genetic analyses have been done
to try and explain virulence, phenotype and host-preferences of these members with no
success. Recent advances in mass spectrometry techniques enable us to analyse thousands of
proteins simultaneously and explore the possible proteomic variation between these members
that could contribute to the phenotypic, virulence and host-specificity characteristics of the
MTBC members. In this study, we aimed to characterize the proteomes of M. bovis and M. caprae by
analysing the high and or low abundance proteins, relative to M. tuberculosis H37Rv, which
could possibly explain virulence mechanisms and host-specificity of these MTBC members.
Whole cell lysate protein extracts were extracted from mid-log phase cultures of
M. tuberculosis H37Rv (A600 = 0.7), M. bovis (A600 = 0.65) and M. caprae (A600 = 0.7).
Proteins were fractionated by SDS-PAGE and in gel reduction/alkylation and trypsin digests
were done. Peptides were identified using LC-MS/MS on the Orbitrap Velos mass
spectrometer and their corresponding proteins were identified by searching peptide databases.
Protein functional groups were assigned according to TubercuList. To provide an integrated
overview of the overall network of protein expression (rather than just limit analysis to
individual proteins), pathway analysis was done on the differentially expressed proteins of M. bovis and M. caprae using PATRIC (Pathosystems Resource Integration Center) and
pathways were visualized using iTUBY (Interactive Pathway Explorer database).
We detected 2199, 2367 and 2350 proteins for M. tuberculosis H37Rv, M. bovis and
M. caprae which correlate to 60% of the proposed M. tuberculosis proteins being expressed
during log-phase. Considering similarities between genomes, it was no surprise that the
functional distribution of the detected proteins extracted was similar. Metabolic pathways
affected by the proteins which were in higher abundance in M. bovis and M. caprae included
amino acid and lipid metabolism, oxidative phosphorylation and xenobiotic degradation. The
over-abundant proteins in M. bovis and M. caprae were also involved in ribosomal proteins
and carbohydrate metabolism, respectively. Lower abundance proteins in these species were
found in lipid and pyrimidine metabolism. These affected pathways can be associated with
the ability of M. bovis and M. caprae to adapt to their environment more readily which helps
them to survive inside the hosts and cause severe pathogenesis. In this study the proteomes of M. tuberculosis H37Rv, M. bovis and M. caprae were
characterized and the variation between detected proteins and protein abundances explored in
order to describe differences between these closely related strains. Future research on animaladapted
Mycobacterial species will address knowledge gaps that are needed to prevent
transmission and spread of the disease. Understanding the mechanisms of virulence and
pathogenicity could lead to development of efficient vaccines and diagnostic tests for a
variety of animal hosts.